Author

Natarajan, Maya

Date

1996

Advisor

McIntire, Larry V.

Degree

Doctor of Philosophy

Abstract

Enhanced, abnormal adherence of sickle erythrocytes to the endothelium lining blood vessels is hypothesized to contribute to vasoocclusion in sickle cell patients, but the specific molecular mechanisms by which these erythrocyte-endothelial interactions occur have not yet been fully elucidated. Alteration of endothelial function, or frank endothelial damage, may also be involved in the pathogenesis of vascular occlusion. In this thesis, these two different aspects of vascular occlusion, namely adhesion and damage, were studied.
A parallel plate flow chamber and computerized phase contrast microscopy coupled to digital image processing were used to visualize and analyze the adhesion of sickle red cells to 1, 4 and 24 hour IL-1$\beta$ stimulated endothelial cells. In addition, damage to activated endothelial cells due to short and long term perfusion of sickle erythrocytes was determined.
Adhesion studies. Pretreatment of monolayers with 50pg/ml of IL-1$\beta$ for 1, 4 and 24 hours caused approximately 16-fold increases in adhesion of sickle cells to activated endothelium at all time points. RGD peptides, enzymes and monoclonal antibodies were used to determine the pathways involved in these sickle erythrocyte-endothelial interactions. Results indicate that there were different endothelial adhesion receptors involved at the different time points: an $\alpha$v$\beta$3 pathway after 1 hour of IL-1$\beta$ stimulation, another pathway, partially mediated by E-selectin, after 4 hours of IL-1$\beta$ stimulation, and a VCAM-1 pathway after prolonged exposure ($>$9 hours) of endothelial cells to IL-1$\beta$. The $\alpha$4$\beta$1 integrin and SLe$\sp{\rm x}$/SLe$\sp{\rm a}$ carbohydrates were established as the sickle cell determinants in these interactions.
Damage studies. Perfusion of sickle cells over 4 and 24 hour IL-1$\beta$ stimulated endothelial monolayers for short periods of time resulted in damage to the endothelium. About 6-8% damage was observed on 4 and 24 hour IL-1$\beta$ stimulated endothelial cells due to the perfusion of sickle cells. In addition, there was a definite correlation between damage and adhesion on the 24 hour activated cells, but this correlation was much weaker on the 4 hour stimulated cells. Perfusion of sickle cells over 4 and 24 hour IL-1$\beta$ activated endothelial monolayers for long periods of time resulted in hemolysis and indicated that modifications to the apparatus were required in order to obtain meaningful results.